JPH03500410A - Purification of monomeric interferon - Google Patents

Abstract

Fast moving monomers of alpha-2 interferon (''IFN-alpha-2'') are purified from slow moving monomers and oligomers of interferon and from noninterferon proteins using a combination of immunoaffinity chromatography, reverse phase HPLC, cation exchange chromatography, and gel filtration. Two fast monomer forms, FMM1 and FMM2, are further distinguished on the basis of charge and hydrophobicity and FMM2 is obtained in essentially homogeneous form with respect to size, hydrophobicity and charge.

Description

[Detailed description of the invention] Monomer interferon purification The present invention provides a novel fully oxidized homogeneous monomeric human leukocyte interferon. Regarding isolation methods. More specifically, this invention provides immunoaffinity chromatography. chromatography, reversed phase chromatography, cation conversion chromatography and The fully oxidized form is purified through a series of purification steps including filtration chromatography. The present invention relates to a method for isolating alpha interferon.

Background of the invention There are two α-2 subspaces of human leukocyte interferon (IFN-α-2). In the intramolecular disulfide @ (CYS’-CYS” and CYS”-CYS13”) It is a 165 amino acid polypeptide chain with a well-characterized secondary structure [ G. Bodo and 1. Pogy.

[Different molecular species in affinity-purified recombinant human interferon α2] Characterization of the Interferon System.

pp. 23-27 (1985); R,%! 1etzel et al., “From Escherichia coli extract Properties of purified human α-interf, on J, J, Interfer R es, 1°, pp. 381-90 (1981)). However, affinity In the produced polypeptide, BO (IO and Pogy are also disulfide Either the bond is mispaired or the essential cysteine is partially or completely removed. We found a species of IFN-α-2 that was reduced. Some of these forms are low Has specific activity [H.

Moorehead et al. [in antiviral activity and funformation stability] The 29-138 disulfide bond of human α-interferon subtype A Role J, Biochemistry, 23.2500-07 (19B4) ]. Alternatively, they may exhibit a different conformation than the native molecule, resulting in may cause adverse reactions when administered. partially or fully redeemed This form is less safe and can result in a more twisted molecule.

The literature describes slow migrating monomers with at least one free sulfhydryl group ( slow-moving monomer; SMM) (partially or completely (reduced form) and a fast-moving monomer with two disulfide bonds v - (fast-rnoving monomer (FMM) (fully oxidized different forms). The free sulfhydryl group of SMM forms an intermolecular disulfide bond. may generate oligomers via These oligomers have a lower ratio CS that has activity and may cause adverse reactions in humans, Purification of Pe5tka and S, J, Tarnowski, r interferon J , Pharmac, Ther., 29.299-319 (1986). ].

Methods for measuring interferon oligomers are known [S.

Pe5tka et al., “Specific for protein dimers, trimers and high oligomers” Immunoassay J, Anal, Biochen+, 132.328-33 Page (19B3); S, Pe5tka et al. “Measurement method for terferon dimer and high oligomer”, Meth, Enzymo 119, pp. 588-93 (1986)].

After extensive purification of the protein, D. R. Thatcher and N. Panayo tatos isolated recombinant IFN-α-2, which they identified as the main component with a pI of 5.9. Characterized as comprising three smaller anodic bands [Recombinant human Purification of IFN-a, Meth', Enzy+nology, 119°16ro1 77 pages (1986)). According to Thatcher and Panayotatos Purification of properly folded and fully oxidized monomers is easy if The reason is that these “conformational deformations” are different from that of the native molecule. This is because they have very similar properties. Additionally, their research shows that According to Conformational deformation may occur due to the reduction potential. this Their observed conformational variants may result from reduced monomers.

In view of the clinical importance of IFN-α-2 in its pure, fully oxidized form, FM Attempts have been made to isolate M from other forms of the protein. To purify IFN Many methods have been described in the literature. European patent application 108.585 is , interference due to prolonged incubation at acidic pH and elevated temperature. Reference is made to the removal of both oligomers and slow monomers from Elon preparations.

A., M., Fel ix et al., “Recombinant human whitening by high performance liquid chromatography” Analysis of various forms of blood cell interferon J, Methods in Enz yrnology, 119. pp. 242-48 (1986), J. porat. [Metal chelate affinity chromatography for protein fractionation] "A New Approach", Nature (ONDON), 258. pp. 598-99. (1975)) from FMM using a modification of the metal chelate chromatography method. reported the separation of M. U.S. Patent 4.432.895 treats redox reagents Conversion of oligomeric interferon to monomeric interferon by It is mentioned that However, separation of well-oxidized fast-migrating monomers The law is not stated. Uniform FMM in its native conformation can be isolated in large quantities. Because of the difficulty of separating, there is still a need for a method of manufacturing it.

Summary of the invention By providing a means to isolate FMM in a substantially pure native form, the present invention Solve the problems mentioned above. More specifically, the present invention provides non-interferrochemical A method for isolating FMM from protein, oligomeric interferon and SMM. provide. Furthermore, the method uses different FMF types (i.e. FMM, and FMM2 ) provide mutual isolation.

According to this method, FMM is purified from other interferons (I) through various purification steps. FN) and non-interferon contaminants. In particular, this method Includes immunoaffinity chromatography followed by reversed phase hepatic LC. FM The two forms of M, FMM and FMM2, have the same size, so 5O They cannot be separated from each other by S-PAGE. However, the inventors based on hydrophobicity and charge, using reversed-phase HPLC and cation-exchange filters, respectively. Separate using chromatography. Following this method, the next step is to gel-filter FMM2. It can be purified to homogeneity by filtration.

Brief description of the drawing Figure 1 shows the fully oxidized form of recombinant α-2 interferon (fast migrating An outline of the purification method for the production of monomer) (FMM, ) is shown.

Figure 2 shows a typical fraction of eluate from immunoaffinity chromatography. Preparation RP-HPLC elution profile (A); FMM fraction from RP-HPLC Cation exchange chromatography elution profile of (B); RP-)IP Second cation exchange chromatography elution profile of FMM fraction from LC (C): and Sephadec of FMM2 from cation exchange chromatography. Figure 3 shows the G-50 gel filtration elution profile (D).

Figure 3 shows the results of increasing the column operating temperature from 25°C to 40°C. Figure 2 shows an improved RP-HPLC separation between FMM, and FMM2.

Figure 4 shows RP-HPLC analysis of recombinant IFM-α-2FMM2 purified intermediate. .

Figure 5 shows intron “’A and recombinant IPM-α-2FMM, and RP of FMM2. - Shows HPLC comparison.

Figure 6 shows R of recombinant α-2FMM2, intron “’A” and Roferon(R)A. P-HPLC' comparison is shown.

FIG. 7 shows trypsin maps of FMM and FMM2.

Specific description of the invention The present invention uses both hydrophobicity differences and charge differences between FMMs, thereby allowing individual Complete separation of the chemical substances to produce pure, stable, and fully oxidized monomers I can do it. Antibody column eluate containing recombinant lFF-α-2 (in the purification step) products of the first stage) are also FMM, slow-migrating monomers, fragments, dimers and It contains highly oligomeric and non-IFN proteins. This protein mixture is mainly The 5 separations Gro up, He5peria, CA), and the protein is a large number of homogeneous (all i.e., by a single solvent concentration) elution step. Next, F Both fractions containing MM are coupled to a cation exchange column. This cation exchange step allows the separation of protein molecules based on differences in net positive charge at specific pH.

FMM isomers are isolated by using homogeneous buffered salt elution. cation exchange Based on their order of elution from the column, these isomers are FMM, and F It is named MM. Perform a buffer exchange and remove any remaining ol from the monomer. A size exclusion column is used as the final step to separate the oligomers. Final purification The obtained FMM was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (S OS-PAGE), isoelectric focusing gel electrophoresis (IBF) and analytical reversed-phase high performance liquid crystal Characterized as a single species in chromatography (RP-HPLC).

The method of the present invention comprises recombinant α-2 interferon and natural α-2 interferon obtained from leukocytes. 2 interferon or α-2 interferon prepared by organic synthesis can be applied to These also include other α-interferon monomers. - It is also useful for obtaining shapes.

In the purification of naturally occurring or genetically engineered (i.e., recombinant) proteins, In addition to possible modifications as a result of the manufacturing process, possible post-translational modifications of the native protein Possibility exists.

These modifications take the form of chemical reactions on the amino acid side chains, e.g. conversion to onine sulfoxide, or amino acid residues such as asparagine and glucose. This will take the form of deamination of tamine. In addition, exopeptidase Removal of terminal (carboxy or amino) residues is a possible mechanism for modification of native proteins. It is.

At least in the case of IFN-α, the purification method of the present invention It allows the recovery of biologically active 1,000-year-olds. Increased purity means stability and reduce the risk of undesired reactions to contaminants and the resulting product. increases the specific activity of

FMM produced according to the method of the invention prepares pharmaceutically useful compositions. can be formulated using known methods. Such compositions are also preferred. or a conventional pharmaceutically acceptable carrier. Also, auxiliary agent , excipients or other agents. The resulting preparation is treated with IFN-α. It will contain an effective amount of FMM for intravenous, parenteral or topical treatment.

The FMM2 of the present invention is processed by proteolytic enzymes or chemical agents to obtain its active fragments. can be selectively processed. This is also shared by polypeptides or other substances. linked by conjugation or other methods to produce larger proteins or protein conjugates. can be done.

FMM purified according to the method of the invention can be used in various forms. good The preferred form depends on the intended mode of administration and therapeutic use. For example, US patent See Nα4.680.175 (Topical Administration Vehicle). Dosage and rate will depend on various factors characteristic of the disease to be treated.

The following examples are included so that the invention may be better understood. These examples are are for illustrative purposes only and are not to be construed as limiting the scope of the invention. Shouldn't.

In this example, we prepared yeast proteins, insulin, from yeast cell lysates. Recombinant essentially free of terferon fragments and abnormal forms of interferon molecules A method for the collection of leukocytes (α) IFNFMM is described.

Expression of recombinant α-2 interferon Co11aborative Research, Inc. (Lexington, MA) American Type Power Luture Collection from (Rockville, Maryland (ATCCNn20663)! Deposited Expression vector C (S281) was used to transform yeast [Saccharomyces cerevisiae (S acct+aromyces cerevisiae), the IFN gene was cloned in l. DNA techniques were used to clone and express.

The stock to be fed to the immunoaffinity column is a fermented product containing yeast protein. It was a cell-free clear cell lysate obtained from. The present inventors have developed a method for homogenization. tion, as well as a combination of centrifugation and tangential microfiltration. Clear cell lysates were obtained by the step method. This gives a clear lysate, which is then treated with - IFN-α2 monoclonal antibody - added to Sepharose column. can get The eluate contains IFN monomers and oligomers, but non-IFN protein contaminants. (e.g., non-specifically adsorbed yeast proteins). Learn more about purification methods The details are as follows.

Immunoaffinity chromatography - The present inventors used genetically engineered yeast collecting and disrupting the cells to release recombinant IFN-α-2; and Unbroken cells and cell debris using centrifugation and tangential microfiltration was removed. We bound the resulting clear cell lysate to Sepharose. An immunoassay comprising an anti-INF-α-2 monoclonal antibody (rL1-1) Added to the affinity matrix. The inventors have discovered that interferon sci. LIT 1 monoclonal antibody was obtained from Nance Co., Ltd., but other anti-α2 itan monoclonal antibodies It could also be replaced by lon antibodies [e.g. US Patent Nu 4.432.1] 47; European patent application Nα91.543; British patent Nα2.111.5 27]. We used cell lysates with our immunoaffinity protein. Recombinant lNN - bind α to the matrix and remove most other cell lysate components and proteins. Removed. We constructed the column with 1,5 M NaCR and 50% (V/V) Washed with phosphate buffer containing ethylene glycol. Eluent paste, 0.28 A second phosphate buffer containing 0.15 M NaC17 until the 0 trace is towards 0. Solution (p) 17.4) was used. After the LITI/S matrix has been thoroughly washed, Bound IFN in 0.1M citric acid (p)1 containing 0.3M NaCj2 2.0). Elute with acetic acid or glycine buffer It may be effective as a drug.

Modifications to the initial treatment of crude cell lysates can replace the entire purification process with immunoaffinity. One step of tea chromatography could be eliminated. For example, crude cell lysis The substances could be batch adsorbed onto a suitable reverse phase or ion exchange medium.

The partially purified IFN is then removed batchwise or column-wise and the IF N can be further purified as follows.

Reverse phase chromatography (RPC) Next, we used RPC to analyze SMM, dimer and high oligomer IFN FMM was separated from non-IFN-miscellaneous proteins. The inventors have developed a high-speed reverse-phase Liquid chromatography (RP-HPLC) with automatic gradient controller, two HPL Waters biner equipped with C pump, LC spectrophotometer and recorder/integrator It was performed on a Waters Binary IIPLC system. C- 4 columns (22mm x 250mm; particle size 10p; pore size 300) was equilibrated with 0.1% (W/W) IJ fluoroacetic acid in water. LITI/S Coupling IFN to the reverse phase column by loading the eluate directly through pump A I let it happen. The bound protein was transferred from the C-4 column at a rate of 18 ml/min using the following procedure. It was eluted with a gram gradient.

1146 linear 41 46 * 45 100 linear Cocote, solution B is 0.1% (V/V) TFA in acetonitrile (CH3CN). It consists of The bound protein is eluted from the C-4 column, collected as separate halves, and then FMM, and adjacent peaks corresponding to FMM2 were purified for further purification. I did a rule.

Now, with respect to Figure 2A, the fraction between 15.5 and 18.0 minutes contains FMM. . Both of these fractions account for a relative area fraction of approximately 25% of the total protein loaded. This special For the given column and operating conditions, FMM and FMM2 have approximately 46% B content. It eluted.

The ratio of FMM and FMM2 was approximately 50:50 based on the relative area ratio. However, this ratio can change to 10:90=FMM, :FMM. 14. The peak eluting at 55 minutes does not contain yeast-derived interferon protein.

In some cases, hydrophobic interaction chromatography can be replaced by RPC, which Therefore, the use of organic solvents can be avoided. In addition, the scale of the reverse phase stage is Column packing materials with larger particle sizes could be used in the column.

This allows the use of lower pressure chromatography, which makes it more expensive. ) avoids the need for IPLC equipment and requires less maintenance; It will become possible to more easily automate the entire system.

Further modifications to the RP-HPLC steps include the use of gradients and/or homogeneous solutions; flow rates; increase or decrease in organic modifiers other than acetonitrile, such as n-propanol, 2-Propertool, methanol, ethanol, dioxane and as an eluent Other substances used; ion pairing agents other than TFA, such as sodium dodecyl sulfate use of phosphoric acid or perhydrochloric acid, or perhydrochloric acid salts; increasing the amount of ion-pairing agent (i.e., greater than or decreased by 0.1% (V/V): with other fixed hydrophobic groups The use of stationary phases, such as C3, Csl, Csl, CI8 or phenyl; and 0 operation at temperatures ranging from 56°C to 56°C. one or more of the above parameters Several modifications are aimed at optimizing the separation between FMM2 and other components. Should.

We tested the effect of varying the particle size of the C-4 filler and The loss of separation between monomer species increases as particle size increases using elution conditions. However, other recombinant α-2 It has been found that separation of monomer from form can still be achieved.

FMM, and FMM2 are based on the hydrophobic differences between these two species, e.g. By varying the ram temperature, separation can be achieved using reverse phase techniques. Figure 3 The column temperature was adjusted at 25 °C (panel A) and 40 °C (panel B). The two chromatograms produced by the method are compared. FMM2 and F at 40℃ MM, the separation achieved at 25°C (peak separation approximately 3 minutes) (peak separation approximately 1 minute).

We tested the S-200 gel filtration stage at this stage; Nevertheless, we decided to use the C-4 column for several reasons: ( 1) A large S-200 column is required; (2) Loading the S-200 column (3) sample must be concentrated before gel filtration; (4) The S-200 column has a limited capacity; (5 ) S-200 Chromatoduram develops gradually (over 24 hours); and (6) Separation of oligomers, monomers and fragments using S-200 column is performed using reversed phase chromatography. This is worse compared to the separation that can be achieved by graphics.

cation chromatography Using cation exchange chromatography, we found FMM and FMM The two monomers were separated based on their net charge difference at pH 5.2.

The present inventors demonstrated that sodium hydroxide of CM-Sepharose CL-6B gel was reduced by ammonia. After exchanging the column counter ions, the cation exchange column was heated with 0.05M ammonium acetate. The mixture was equilibrated with Ni (pH 5.2). Acetonitrile and trichloride from RPC Although fluoroacetic acid can be separated by dialysis, phase separation or lyophilization, FM Preferably, the organic fraction containing M is diluted.

Both aliquots were diluted approximately 8 times with 0.05M ammonium acetate (pt15.2), and then at a linear velocity of 25 marks/hour onto a CM-Sepharose CL-6B column equilibrated with Loaded directly. Next, add approximately 2 column volumes of 0.05M ammonium acetate to the column. (pH 5.2) and then 0.15M ammonium acetate (pH 5.2). -2>, it was eluted at a linear velocity of 9.0 CII+/h. Separated FMM, and After collecting both parts of the FMM2 monomer peak, 1.0 M ammonium acetate (pH 6,0) at a linear velocity of 25 cm/h to elute the strongly bound oligomers. Ta. Using a salt gradient elution of 0.2M-0.3M ammonium acetate (pH 5,0) However, especially for large-scale production, the above-mentioned homogeneous salt elution is preferred.

Regarding Figure 2B, the FMM peak elutes at 4-5 column volumes, while the PM M2 pinic elutes at approximately 5.5-7 column volumes.

One step of cation exchange chromatography is to increase or decrease the linear velocity. ammonium acetate; by changing the pH and/or ionic strength of the eluent; other buffers or counterions, such as sodium succinate or sodium acetate. By using column geometry; By changing the column geometry; By operating at temperatures (up to 50°C); using gradient elution or homogeneous elution by; ion exchange matrices with different ligands or ligand densities; For example, CM-Sepharose-Fast Flow, SP-Sephadex, CM- Using Sephadex, CM-) Lisacrylic M or 5P-) Lisacrylic M or by a support matrix that is mechanically stronger than Sostgel Sepharose. By using a gas, for example CM-Fast Flow or CM-Silica, Can be changed. Needless to say, FMM.

and other components, especially FMM, as shown in this example. Any changes to the method described may be introduced.

gel filtration chromatography Next, we ultrafiltered the FMM2 peak from the CM-Sepharose column. It was concentrated to a protein concentration of about 2/-.

This was equilibrated with 0.05M succinate buffer (pH 5,0) 2,5 cIuX 80C1l + gel filtration column (Sephadex G-50 Superfa In). Next, we used succinate buffer to It eluted at linear velocity. Succinate buffer is an acceptable excipient for parenteral administration. This is because it is a drug.

Regarding the second diagram, the two separated peaks emerging from the column are each a dimer. - and FMM2 are shown. FMM, collect peaks and unbind 0.22-pores Pass through the filter and add 0.55M succinate buffer (pH 5.0) to a width of about 0.5 to 1. ．． A purified drug concentrate with a protein concentration of 0 mg/ml was obtained. The inventors Observe that the FMM2 of the filtration eluate is homogeneous on a silver-stained 5O3-PAGE gel. I guessed it.

Any gel filtration column that will fractionate in the appropriate molecular weight range, such as 5e phacryl S-2005upperfine, 5uperose-12゜ FractoHel 50S, Llltrogel ACA 54 and TSK -250 gel filtration matrix can be used at this stage. In addition, gel Ultrafiltration can be used in place of the filtration step to speed up the operation. deer However, since ultrafiltration does not seem to remove all remaining dimers, filtration is preferred. Finally, for each step, increase the flow rate to It can save time.

Table 1 shows the results for recombinant 1FN-α-2FMM, protein starting from antibody column elution. This is a summary of the recovery rate and yield per purification step. Overall recovery of material is 5.8%. FMM in monoclonal antibody column eluate, relative to content The recovery rate is 48.6%.

Reversed phase chromatography 65 24.4 24.4 Concentration 17.9 6.7 88.2 ” Estimated by optical density at 280 nm.

Next, in order to determine the specific activity of the purified FMM of the present invention, we , Rubenstein et al. our own of Virology, pp. 37.755-58 (1981) Cytopathic effect assay (CPE) was performed using the method described in [1]. First, the interface Cells known to be sensitive to cellulose should be titered. Incubated with serial dilutions of the sample. Each dilution will challenge added. Viral cytopathic effects on cells after re-incubation We observed dilution levels that achieved a 50% reduction in . Concentration expressed in standard units International standards, such as the US National Institutes of Health (National Institutes of Health) s　of　leath) established by Yasuda, Maryland, This was determined by simultaneously conducting assays for

We simply use immunoaffinity (i.e., LITI/S) chromatography. Approximately 1°0 ■Compared to protein, the specific activity of FMM2 is approximately 2.3 x 10” units/■ It was discovered that it is a protein.

Example■ In this example, we simplified the cell lysate “clarification” method and used only low-speed centrifugation. did. As a result, the immunoaffinity column feedstock produces a cloudy cell solution. It was a decomposition. The inventors used immunoaffinity to process the resulting cell lysate. A fluidized bed reactor for nitty chromatography was used. We have developed a three-part homogeneous An elution program was included for the RPC step. This is the resulting LITI/S eluate contains substantial amounts (up to 35%) of non-IFN protein contaminants, such as yeast proteins. Because it was. The first step essentially removes the interfering yeast proteins; the second step Dissolve IPN FMM for further purification by ion exchange chromatography. and a third step removes large amounts of IFNSMM oligomers and fragments.

Recombinant JPN-α-2 in crude cell lysate after treatment in LITI/S fluid bed Approximately 600 μl of recombinant IFN-α-2, representing approximately 50% of the . The LITI/S eluate (7,2A) was filtered through a 0,45 micron membrane and 8 times onto a preparative Vydac C-4<22mmX250own) reverse phase column. Loaded separately. Each load was applied using a multi-stage uniform gradient of acetonitrile as shown below. It eluted.

Time (minutes) B (%) Curve 1143 linear 20 43 * 2146 linear 36 46 * 40 100 linear 45 100 * FMM fractions representing approximately 24.5-26.0 minutes of elution from each run were pooled. . Represents 92% of the theoretical amount of 115 ■ as determined by analytical RP-HPLC. 105 μm of FMM was recovered from the LITI/S column eluate.

The FMM fraction was diluted to 6% (V/V ) in acetonitrile and 200 ml (4,4X14 cm) of cation exchanger. The mixture was loaded onto a conversion column (CM-Sepharose CL-6B). This column is 0.14 Isocratically with M ammonium acetate (pH 5,2) ) eluted. The results are shown in FIG. 2C. Molar concentration of ammonium acetate buffer A slight decrease (from 0.15M to 0.14M) was observed in the previous separation (see example work). ), the separation between FMM and FMM2 was improved. Both indicating FMM2 are combined and these are filtered using ultrafiltration to produce approximately 5 μg of protein/rn1. concentrated in did. Sephadex G-50 superfine gel filtration of concentrated FMM2 Load the column to remove traces of oligomer and add ammonium acetate and 0. 05M succinate buffer (pt15.0). P from gel filtration column The protein concentration of the MM2 eluate is approximately 1 mg/mf, and approximately 2.2 It had a protein specific activity of ``unit/■.'' Table 2 shows the details for FMM2. The recovery rate and yield for each production stage are summarized. Overall yield based on protein is from total protein 7.3%, based on the total FMM2 contained in the LITI/S column eluate. It was about 50%.

Table 2 LITI monoku Local antibody 600 100 100 116 Figure 4 shows each intermediate in the purification process. shows a comparison of analytical RP-HPLC profiles for and FMM2 This ensures the homogeneity of the final purified drug concentrate.

Example■ The characteristics of FMM and FMM2 are We prepared samples of FMM2 as described above for characterization.

To prepare FMM, cation exchange FMM and fractions were analyzed by RP-HPLC. Re-chromatograph and collect all peaks corresponding to FMM. The samples were further purified by removing residual FMM2 or SMM. both Minute-by-minute analysis revealed that the specific activity of FMM was approximately 200 x 10 1'U/■) was found to be approximately the same.

Analytical isoelectric focusing J LKB Application Note 25 0 (1977)]. . Diafificate the sample against 5 mM Tris-HCA (pH 8,0). 10. Amicon Sen equipped with OOOMWCO membrane Concentrated in an Amicon Centricon tube. Hiramae molding LKB Ampholine (Amphol 1ne) RPG plate (p )13.5~9.5) using LKB (Bromma, Sweden) minutes The isoelectric point was measured using an isoelectric point device for analysis. Approximately 10ha (approximately 5-10.q protein) Prior to application, the gel was pre-focused at a constant power of 50 watts for 0.5 hours. I clicked. Incubate the electrophoresis with cooling for 1-2 hours or until a constant current is achieved. It was pneumophoresed. Pharmacia Fine Chemicals (Piscataway, N. Isoelectric point markers were obtained from J). Gel at room temperature, 30% methanol, 3.5% Fixed in sulfosalicylic acid, 11.5% trichloroacetic acid solution. Next, apply the gel 0.1% (w/v) coma in 8% acetic acid in 25% methanol for 10 min at 60°C. It was stained using See Brilliant Blue R-250. Finally, 25% methano Destain to a clear background by diffusion in a solution of 8% acetic acid in a Ta.

The results of IEF gel electrophoresis show that FMM has a pr of about 5.85, while FM It was shown that that of M2 was about 6.09.

The present inventors found that the difference in isoelectric point between FMM+ and FMM2 is due to methionine sulfoxide residues. I don't believe it can be attributed to. Because these two types of brome This is because undigested peptide maps cannot be distinguished.

Dynamic (SO3-PAGE) We prepared our sample with 5mM Tris-)1(J' (pH 8,3) Among them, U, Laemmli (r during the assembly of the head of bacteriophage T4) Cleavage of structural proteins J, Nature (London), 227°680-8 5 (1970)]. Contains 5-10jIg of protein A volume of concentrate was mixed with a volume of sample buffer to make 10mM Tris. -0.1% (Ill/V) SO3°10% (ν/V) in HCl (pH 6,8) ) The final concentration of glycerol was obtained. The sample was then heated to 95°C for 3 minutes.

If a reduced sample is required, add 2-mercaptoethanol to the sample. to give a final concentration of 10% (V/V). The sample was coated with 4% polyacrylamide. into preformed wells in tacking gel (1, OX, 18x O, 75 cm). and apply them to 14. each containing 0.1% (MV) of SDS. Electrophoresis on 5% polyacrylamide separating gel (16X18XO, 75cJn) did. Electrophoresis was performed at a constant current of 30 milliamps, with color tracking while cooling. Continue until the bare tip (bromophenol group) migrates to the bottom of the gel (about 3 hours). Ta. Fix the gel and add isopropanol/vinegar M/water (2 ml) for 1 hour at room temperature. 5: 10: 65) Medium 0.1% (W/v) Coomassie Brilliant Blue R -250 and using methanol/acetic acid/water (5:10:85). It was destained to obtain a clear background by diffusion. Sensitivity of dyeing process If the protein loaded on the gel needs to be stained with Coomassie blue, If the detection limit is lower than the detection limit according to the method of Merr et al. Simplified protein detection and image enhancement method in gels J, Electrop horesis, 3.17-23 pages 982)] 0.012M nitric acid The color was washed with a silver (AgNos) solution. Using 5O3-PAGE analysis, the present invention They found that PMM and FMM2 under both non-reducing and reducing conditions. I found that it was not possible to tell the difference. Under non-reducing conditions, both are about 19,000 Dal It migrated with an apparent molecular weight of tons.

C. Amino acid composition The inventors are J. W. Eveleigh and G. D. Winter (r. Determination of acid composition J, Protein 5equence Determina tion, pp. 91-95 (1970)) at 100°C for 24 hours. time, to hydrolyze 0.5-1.0 nmoles of sample under standard conditions. The concentration of amino acid residues was determined using hydrochloric acid. For determination of MET and CYS concentrations For this purpose, performic acid hydrolysis was performed. Durram the amino acids of the resulting hydrolyzate. Using a Model D-500 amino acid analyzer, The analysis was performed using fluorescent light and fluorescence.

A total of 8 samples each of FMM2 and FMM were analyzed. mean and standard deviation are shown in Table 3. This result shows that between FMM2 and FMM There are no significant differences based on amino acid composition.

Table 3 Amino acid analysis (± standard deviation) Residue FMM, FMM2 Estimated value (0) ASX 13.9N±1.05> 14.22 (+1.20) 12THR101010 GLY 7.44 (+1.10) 8.02 (+1.87) 5ALA 8.8 6 (+0.53) 8.9N±0.69) 8VAL 6.61 (+0.36) 6.40 (+0.21>7HIS 2.93 (+0.53) 3.05 ( +0.63) 3ARG 10.47 (+0.82) 10.66 (+0.9 5) 10TYR5,20 (+0.09) 5.17 (+0.16) 5PHE 9.50±1.37) 9.67(+1.09) 10SER13,89(+ 0.55), 14.15 (+0.50) 14GLX 28.06 (+0.8 2) 28.16+1.07) 26ILE 7.39 (+0.61) 7.4 8 (+0.68) 8L E U 20.47 (+1.83) 20.76 <±2.46) 21LY3 9.97 (+0.89) 10.04 (+1.2 0) 10PR04,99 (+0.54) 4.46 (+1.26) 5CY S 4.00 (+0.85) 3.85 (+0.49) 4MET 5.35 ( +0.35) 5.35 (+0.21) 5TRP 2 ('') 2 ('') 2 D, FMM, and FM), + 0.5 to 1.0 nmole of N-terminal sequence of 2 The S-carboxymethylated sample was dissolved in TFA and the current automated ed. Mann decomposition method [M, W, Hunkapiller, E, Hood, r ultra-high sensitivity "Analysis of phenylthiohydantoin by high performance liquid chromatography", Me thods in Enzyrnology, 91.486-93 (198 3)] using a 470A gas phase sequencer (Applied Biosyst ms). PTH-amino acid with WISP automatic sampler and RP-HP with a Waters Model 6000 gradient system with a fixed wavelength detector. Analyzed using LC. Nelson Analytical Software Data were analyzed using an IBM computer.

FMM2 results CM, CYS-ASP-LEU-PRO-GLN-(THR)-HIS-5 ER-LE [l-GLYSER-ARG-ARG-(TIIR)-LE Low ME The N-terminal sequence of T-LEU-LE low ALA-GINFMM2 is mature recombinant α-2. CD, V. Goeddel et al., “Eight different published information about 1FN. Structure of cloned human leukocyte interferon cDNA J, Nature, 290. pp. 20-26 (1981): Corresponds exactly to l, with the exception that Goedd el estimates that LYS ranks 23rd. Amino acid residues enclosed in parentheses are those of the present invention. indicates uncertainty in the person's analysis.

FMM, results Sequence data could not be obtained using the conventional Edman decomposition method. Therefore, The inventor concluded that the protein was blocked at its N-terminus.

E, determination of sulfhydryl group We investigated the sulfhydryl groups in pure preparations of FMM, and FMM2. ElliIIIan reagent 5.5'-dithiobis-(2-nitrobenzoin) Habeeb method [A, P, S.

A, tlabeeb, r reaction of protein sulfhydryl group with Elliman's reagent", Me thods in Enzymology, pp. 25.457-64 (1972 )] was measured by a modified method. 6M Go-HC containing 20mM BDTA j! Solution (pH 8, 3) 666m, 0.5M Tris-HC of 327IJ1 A (pH 8.3, 20rnM EDTA) Sample protein with a width of approximately 10 nmoles , and 90p1 of 10rnlil DTNB in water were mixed gently for 30 minutes. . A freshly prepared cysteine solution in 20mM EDTA (pH 8,3) was used as a standard. The absorbance at 412nrn was measured using the solution (2mM). Under these conditions The extinction coefficient is 13.000-13.600, which is different from the reported value. It was a good match.

These results for PMM and FMM are less than 0.1 mole per mole of protein. Both the FMM of the present invention and FMM2 are fully oxidized. It was shown that

Reversed phase high performance liquid chromatography (RP-HPLC) automatic data controller for F0 analysis controller model 680.2 model 510 pumps, LC spectrophotometer model 481, Rheodyne injector, Waters column temperature control module and Waters who wants to install an integrator)Binary)IPLc RP-HPLC was performed on the System.

The present inventors used a Vydac C-4 reverse phase column maintained at a constant temperature of 40°C ( 4,6n+mX25cm; 5jnn particle size) 1H samples were analyzed. Column was 0.1% (v/v) trifluoroacetic acid in water. (TFA). Follow the following program, 0.1% (V/V) T F　A middle-r ce) = 　)! 1r with Jru(CH3CN) (solvent B) A gradient elution was developed with a flow rate of n1/min.

1545 linear 45 45 + 50 100 linear We monitored the column eluate for absorbance at 280 nm.

Under these chromatographic conditions, we found that FMM, and FMM, each for 27.6 minutes (see Figure 4, step 5) and 30.4 minutes, respectively. run as a single symmetrical peak with a retention time of 10 min (data not shown). observed.

in Enzymology, Vol, XI, pp. 231-39 (1967) ). Approximately 2 nanomoles of 5-carboxyamidomethylated The purified trypsin:protein (FMM, or FMM ) was digested twice. This digestion consisted of 0.1 MNt (4HCOs (pH 8,0) The test was carried out for 2 hours at 37°C. After digestion, the pH of the peptide mixture was adjusted to 2% T in water. It was adjusted to 2.0 by FA.

The peptide mixture was dried and redissolved in 0.1% TFA in water and again Dry. Finally, the peptide mixture was placed in a known volume of 0.1% aqueous TFA.

The present inventors created a trypsin map using RP-11PLc. Approximately 2 nano grams of each digestate separately in 95% water, 5% acetonitrile (0.1% TFA). A reverse phase column (Vydac C-18, 5 micron particle size, 30 0 manhole size, 4.6×250M) were loaded separately. Peptide at 30℃ , 5-50% acetonyl IJ at 0.23%/min.

The peak was motored at 215 nm.

As a control, (a) using the same conditions as used for the peptide mixture; Lipsin only; (b) undigested FMM or FMM2; and (C) alkyl undigested FMM+ or FMM2. Contrast as shown in Figure 7. It was found that the treatment did not disturb the peptide map. The present inventor is FMM, A clear difference between the monomer and FMM2 monomer was observed (see Figure 7). and).

The inventors obtained 1ntron A and Roferon A from a local pharmacy. , and analyzed the interferon composition of each.

This is for determining recombinant leukocyte interferon alpha-2 in complex mixtures. was achieved using a rapid measurement method. This measurement is performed using a tandem column on a reversed-phase HPLC column. C L, R relies on the use of a high-speed monoclonal affinity column coupled to Ybacek et al., “Rapid Dual Column Block of Recombinant Leukocyte Interferon-α-2” 7 Chromatography Measurement J, J, Chromatography, 397.35 5-64 (1987)).

After collecting interferon from this rapid measurement system, H3A-free 1ntron A or Roferon A on an analytical Vydac C-4 column (4, 6mm x 250mm; particle size 5 microns) and 0.1% Chromatogram by stepwise gradient to 44% acetonitrile in trifluoroacetic acid. was developed. Regarding FIG. 5A, the inventors found that Intron A lasted for 16.7 minutes. It was observed to elute as one peak with a retention time. Next, recombinant I A mixture of FN-α-2-FMM and FMM2 was chromatographed under the same conditions. I understood. FMM2 elutes at a retention time close to Intron A (16.8 minutes). , while FMM eluted in 17.3 minutes with a delay of 1 minute (Figure 5B). even more In the study, the present inventors demonstrated that Intron A and recombinant JFN-a-2FAI M, and an equal mixture of Intron AL recombinant IFN-α~2 FMM2. A mixture of equal volumes of was subjected to reverse phase chromatography as described above. Regarding Figure 5C, FM M, and Intron A were co-eluted from the reverse phase column, while PMM l and I ntron A showed each unique retention time (Fig. 5D).

In another experiment, Roferon A, Intron A and recombinant JFN The reversed phase chromatograms of -α-2FMM2 were compared. Regarding Figure 6, each sample The main peak of Roferon A ( Figure 6C) contained a vestigial shoulder on the main peak. These results are similar to Rofer on A is more heterogeneous or that it is recombinant a2 FMM2 (panel Contains more degradation products than either A) or Intron A (panel B) suggest that you are doing so.

The inventor also used isoelectric focusing gel electrophoresis to detect recombinant IFN-a-2FMM2. Comparison was made with Intron A and Roferon A. The results show that According to the study, recombinant lFF-α-2FMM2 had the least isoelectric point species. In Tron A exhibits at least two types of isoelectric point shapes, and Roferon A exhibits at least two types of isoelectric point shapes. At least four types were shown, whereas FMM2 of the present invention showed only one form. Furthermore, Ro The feron AO main band has a different isoelectric point from FMM2 and Intron A. Was. This difference is due to the substitution of lysine for arginine at position 23 of the polypeptide. This can be attributed to a certain difference in the primary structure between α2 and αA.

In other studies, the inventors determined that the original After 5DS-polyacrylamide gel electrophoresis, FMM was transferred to 1ltron A and Compared with Roferon A. This result shows that Intron A contains traces of dimer and Roferon A contains some unidentified They were indistinguishable in size, except that they contained high molecular weight proteins.

Example■ Stability of purified concentrate of recombinant-JPNα2 FMM, recombinant IFN-α-2 A purified concentrate of FMM2 (specific activity 2.3 x 10"U/mg) was added to 0.05 0.48 mg/- in M citrate buffer (pH 5,0) at several temperatures (-2 0°C, 4°C, 25°C and 37°C) for 4 weeks. The inventors believe that C.P. for activity by E assay and for degradation by 5DS-PAGE. Each temperature was evaluated. Specific activity did not change even after 4 weeks at 37°C (average 2.5 x 10’U/■). Furthermore, after 7 weeks at 37°C, the Analysis of FMM2 by 5O3-PAGE shows that oligomers are not detected in non-reduced samples. or exhibit no low molecular weight degradation products and no trace amounts in the reduced sample. Low molecular weight substances were present.

Although the present inventors have shown many specific examples of the present invention, the basic structure of the present invention has been changed. It is possible to provide other embodiments of using the methods and compositions of the present invention. it is obvious. Therefore, the scope of the present invention does not fall within the scope of the present invention. shall be determined by the claims appended hereto and not by the claims appended hereto. Ru.

F/θ/ Purification process steps for producing recombinant α2FMM2! : Yeast fermentation Step 2: Immunoaffinity chromatography am-x,’ cation Exchange chromatography 1st step 5: Gelsi filtration chromatography FI6.2 (A) Reversed phase chromatography Time (minutes) Cation exchange chromatography Example 1 Example 2 F/θ　2　72　Column capacity F/63 Panel A: 25°C Panel B: H)'C sample: url/s eluent Temperature: Adjusted by Waters TCM gradient program Eluent A: 欓gua, yzry Eluent B: CA/3 (run 0./, yaf 5 linear f5 45 45 4f linear jθ　gθ0 55/θ0 F/θ4 Time (minutes) FIG5 Panel A: Panel B; In1rOn A FMM+ and FMM2 (Di compound time (minutes) Time (minutes) FIG6 Time (minutes) FIG.? international search report

Claims (21)

[Claims] 1. A slowly migrating partially or fully reduced form of leukocyte interferon Monomers (SMM) and oligomers as well as non-mono-interferon proteins are substantially The fast-moving fully oxidized form of human α-2 interferon, which does not contain a) a fully oxidized form of human monomer (FMM) comprising: Crude human alpha-2 containing fast migrating monomers of alpha-2 interferon Interferon (IFN-α-2) was loaded onto a low alkyl reverse phase column and Elution; b) loading and eluting the product of step a) on a cation exchange column; c) loading and eluting the product of step (b) onto a gel filtration column; and d) recovering said fast migrating monomer in purified form; Law. 2. IFN-α-2 was first purified by immunoaffinity chromatography. 2. The method according to claim 1. 3. IFN-α-2 was dissolved from the immunoaffinity column using an acidic buffer. 3. The method according to claim 2, wherein: 4. The reverse phase column is a C-4 column, and the FMM is 0.1% trifed. 2. The method of claim 1, eluting with fluoroacetic acid and 46% CH3CN. 5. Claim 1, wherein the cation exchange column is an immobilized carboxymethyl column. The method described in. 6. The FMM is removed from the gel filtration column from about 0.45 portion of the total column volume. 2. The method of claim 1, wherein the method begins collecting. 7. Said reverse phase chromatography of step (a) is carried out on a C-4 column and step (a) is carried out on a C-4 column; The ion exchange chromatography of b) is carried out on a carboxymethylated column. , the eluent of step (a) is trifluoroacetic acid in acetonitrile, and step 2. The method of claim 1, wherein (b) is ammonium acetate. 8. 2. A method according to claim 1, wherein the elution in step (a) is carried out under homogeneous conditions. 9. Different FMMs of α-2 interferon are separated based on charge and hydrophobicity. 2. The method of claim 1, wherein: 10. A partially or fully reduced form of α-interferon, α-interferon - Slowly migrating monomers and oligomers of ferons and non-interferons A fully disulfide-bonded form of α-interface that is essentially free of protein Ron. 11. 10. The interferon is human alpha-2 interferon. FMM described in. The F according to claim 10, which has a specific activity of 12.1 x 10 units/mg or more. M.M. 13. FMM according to claim 10 in combination with one or more other polypeptides A pharmaceutical composition comprising. 14. α-interface comprising a pharmaceutically effective amount according to claim 10. Composition for the treatment of conditions susceptible to lon treatment. 15. A pharmaceutically effective amount of FMM according to claim 10 together with a suitable carrier. A topical composition comprising. 16. A partially or fully reduced form of α-interferon, α-interferon - Slowly migrating monomers and oligomers of ferons and non-intronic proteins substantially free of disulfide, produced by the method of claim 1, essentially free of Human alpha-2 interferon (FMM) in conjugated form. 17. First, IFN-α-2 was isolated by immunoaffinity chromatography. The FMM according to claim 16, which is produced by fractional purification. 18. In step (a) IFN-α-2 is loaded onto a C-4 reversed phase column and a Elute with trifluoroacetic acid in setonitrile, advance the eluate and step (b) ) and loaded it onto a carboxymethylated column and ammonium acetate. 17. The FMM according to claim 16, which is produced by eluting the FMM. 19. From the gel filtration column, we started collecting about 0.45 of the total column volume. 17. The FMM according to claim 16. 20. Different FMs of α-2 interferon separated on the basis of charge and hydrophobicity M. 21. Separated monomers FMM1 and FMM2 and fragments thereof.